PROJECT SUMMARY Stem cells are responsible for homeostasis and repair of most of the tissues in the body. Many populations of stem cells persist in a quiescent state until stimulated to enter the cell cycle, proliferate, and differentiate into functional cells of the particular tissue. In recent years, work from our group and others has drawn attention to several unexpected characteristics of quiescent muscle stem cells (MuSCs), many of which are shared by other quiescent stem cell populations. These include the active maintenance of cellular quiescence, unique metabolic and energetic mechanism in quiescent and activating stem cells, and the presence of large numbers of transcripts for which no protein product is detected. This latter observation raises three major questions that are the focus of this proposal: 1) Is the transcriptional profile of MuSCs (or any other stem cell population) in vivo similar to that of cells that have been isolated and purified by fluorescence-activated cell sorting (FACS)? 2) What are the post-transcriptional mechanisms that are responsible for the absence of protein products when transcripts are present in the quiescent cells? 3) What are consequences of accumulation of those protein products in quiescent cells that necessitate mechanisms to prevent such an accumulation? To address these issues, this proposal is divided into three Specific Aims as follows. Aim 1: To study the dynamics of the quiescent and activating MuSC transcriptome. We will use methods to label nascent RNA in vivo (using 4-thiouracil (TU) and 5-ethynyl uridine (EU)) followed by labelled transcript purification and RNA- seq to assess MuSC transcript dynamics in vivo and ex vivo. We will also profile transcripts using RNA-seq of fixed MuSCs to assess transcript abundance in vivo. Aim 2: To study the translatome and proteome of MuSCs in vivo and ex vivo. We will isolate ribosome-associated transcripts (using the RiboTag mouse) followed by RNA-seq and OP-puromycin labelling of labelled proteins followed by mass spectrometry to assess transcripts that are associated with the polyribosome and translated into detectable proteins in quiescent MuSCs in vivo and ex vivo. We will also assess protein translation in MuSCs in vivo and ex vivo during the process of MuSC activation. Aim 3: To understand the regulation of MyoD translation and the consequences of aberrant MyoD protein expression in quiescent MuSCs. Based on Preliminary Data, we will test the hypothesis that Staufen1 suppresses the translation of the MyoD transcript in quiescent cells, and we will analyze the functional consequences of MyoD expression in quiescent MuSCs by inhibiting those suppressive mechanisms genetically and using an siRNA approach. The overall goals of these studies are to provide a more accurate assessment of the in vivo state of the quiescent stem cell and to understand in greater detail the molecular mechanisms that maintain stem cell quiescence and at the same time prime the cell for activation and differentiation.